Saturday, August 16, 2008

INTRODUCTION

Tables 6-1, 6-2, and 6-3 summarize currently available recommended test methods for the detection of bacterial, fungal, and parasitic pathogens from human specimens. Methods for detection of viral pathogens are discussed later in this chapter. For completeness, the tables include pathogens common to both normal and immunocompromised hosts. Selected specimen collection procedures are shown in Table 6-4. However, this important step in the diagnostic-testing process cannot be overstated. An adequate amount of specimen (using sterile techniques when appropriate) should be placed in the appropriate transport device, and the specimen should be transported to the laboratory under appropriate environmental conditions and within a reasonable period. Because a large number of transport devices are available, consultation with the laboratory personnel is important, especially when unusual bacteria are considered. Comprehensive reviews of the specifics of specimen collection, transport, and storage are described elsewhere. As a general point, specimens requiring strictly anaerobic conditions for viability must be sent in appropriate anaerobic transport devices. Depending on the volume, most other specimens can be sent in either a swab transport device (the swab is immersed in nutrient broth) or a sterile container. Some studies have demonstrated the utility of blood culture bottles for transporting as well as culturing sterile body fluids, especially peritoneal fluid.

RAPID, DIRECT TEST METHODS & CULTURE-BASED METHODS

Introduction

Direct, rapid diagnostic-test methods if available should be used in addition to conventional culturing techniques for diagnosing bacterial or fungal infections in critically ill patients. Most parasitic infections can be diagnosed by direct staining methods, and these tests should be performed emergently, especially if life-threatening infections like malaria are suspected. Rapid diagnostic tests are provided in Table 6-1, and culture-based diagnostic tests, which require longer time periods to complete, are presented in Table 6-2.

Blood

Both direct and culture-based techniques may be useful for identifying common and uncommon bacterial, fungal, and parasitic pathogens in blood. Some of the specialized testing methods shown in Tables 6-1 and 6-2 are generally available only at reference laboratories. If the clinician feels strongly that one of these tests is useful, an aliquot of the specimen should be sent to a reference laboratory. Direct test methods for the detection of bacterial or fungal pathogens in blood are limited in the number of different bacteria that they can detect and are less sensitive than culture methods. Bacterial-antigen tests for commonly encountered encapsulated organisms (Haemophilus influenzae type b, Neisseria meningitidis, or Streptococcus pneumoniae) are easy to perform and available in most laboratories. A latex agglutination procedure can be used to detect the yeast Cryptococcus neoformans.

Blood cultures should be obtained from all patients in whom sepsis is considered. The standard blood culture set for adults consists of 20-30 ml of blood equally distributed between two or among three culture receptacles. Blood volume for pediatric patients is less and is dependent on patient weight. When 20 ml of blood is drawn, the standard practice is to culture 10 ml in an aerobic atmosphere and 10 ml in an anaerobic atmosphere. If 30 ml of blood is drawn, the same practice is followed except that the additional 10 ml is cultured under aerobic conditions. In patients with suspected endocarditis or endovascular infections, conditions in which bacteremia is continuous, two or three separate blood cultures collected at various intervals over a 24-h period are sufficient. For other types of bacteremia, 99% will be detected by three separate blood cultures collected at various intervals over a 24-h period. Most conventional broth-based blood culture systems also have the ability to detect candidemias. Specialized broth or procedures (see lysis centrifugation below) may be required to detect fastidious bacteria and dimorphic fungi, especially Histoplasma capsulatum.

Recently some authorities have questioned whether anaerobic blood cultures should be performed routinely in all patients, especially those in whom anaerobic bacteremia is unlikely. However, it has recently been demonstrated that anaerobic blood cultures may recover some facultatively anaerobic bacteria (eg, Enterococcus spp. and viridans streptococci) more efficiently than aerobic blood cultures. The lysis centrifugation method is a specialized blood culture method which is useful for recovering common as well as many unusual bacterial pathogens and fungi. In this system, blood is inoculated into a test tube containing saponin, a chemical that lyses erythrocytes and leukocytes, thereby releasing intracellular bacteria. The tube is centrifuged, and the sediment is inoculated onto solid agar plates or into broth. Mycobacterium spp., especially Mycobacterium avium-intracellulare, occasionally produce bacteremias in severely immunocompromised patients. The inoculation of the sediment from a lysis centrifugation blood culture tube onto specialized mycobacteria agar or into BACTEC 13A broth bottles is useful for recovering mycobacteria. Controversy exists as to whether blood cultures can be obtained from intravascular lines. Several investigators have shown that such a practice results in the isolation of more contaminating microorganisms than if blood is obtained from peripheral veins. Whenever possible, blood for culturing should be obtained from peripheral veins; however, this recommendation must be considered in the context of the clinical situation of the patients. For example, phlebotomy via peripheral veins may be risky in severely thrombocytopenic bone marrow transplant patients.

Parasitemias caused by malarial or filarial organisms are diagnosed with thick and thin blood smears. Thick smears permit screening for malaria parasites, and speciation is possible by careful evaluation of thin smears. When parasitemias are suspected, the laboratory director should be notified so that proper processing and careful evaluation of blood specimens are undertaken. In some cases, referral of specimens to reference laboratories may be beneficial.

Other Sterile Body Fluids or Tissues

Direct antigen testing for H influenzae type b, N meningitidis, S pneumoniae, group B streptococci, E coli, and C neoformans can be performed on cerebrospinal fluid (CSF) or urine. Direct antigen testing for Legionella pneumophila and H capsulatum can be performed on urine. For staining and culturing methods, CSF or joint, peritoneal, or pleural fluids should be concentrated by filtering or centrifugation. In contrast, Gram stains of urine should be performed on unconcentrated specimens. The presence of =2 bacteria per oil immersion field (×1000) in a Gram-stained smear of a drop of unconcentrated urine should represent ~ 105 colony forming units of bacteria/ml of urine. Other rapid screening tests for bacteruria are commercially available. These tests, which detect either bacteria or leukocytes by direct or indirect methods, are generally no more accurate than the Gram stain method and may be more costly.

Cultures of bone marrow specimens may be particularly valuable for diagnosing Salmonella typhi (the agent of typhoid fever), Brucella spp., disseminated mycobacteria infections (M avium-intracellulare) or H capsulatum and can be processed by using the lysis centrifugation method. Granulomas surrounding small vessels in a bone marrow biopsy (ring granulomas) are associated with Coxiella burnetti (agent of Q fever) infection. Bone marrow specimens may be positive by staining methods in patients with disseminated infection caused by a variety of organisms, including H capsulatum, M avium-intracellulare, Trypanosoma spp. or Leishmania spp.

Respiratory Tract Specimens

All spontaneously produced sputa that are submitted for general bacteria culture should be screened for the presence of squamous epithelial cells. It has been demonstrated that expectorated sputum samples having > 25 squamous epithelial cells per low-power microscopic field are unacceptable for bacterial culture, because these samples likely are contaminated by oropharyngeal secretions. Common community-acquired respiratory bacterial pathogens include S pneumoniae, Streptococcus pyogenes (Lancefield group A ß-hemolytic streptococci), Klebsiella pneumoniae, S aureus, Legionella spp., Chlamydia pneumoniae, and Mycoplasma pneumoniae. Gram-negative bacilli, especially Enterobacteriaceae and Pseudomonas spp., and VRE and staphylococci may cause nosocomial respiratory infection. Pseudomonas aeruginosa and B cepacia are frequently associated with pulmonary infection in patients with cystic fibrosis. All of the above bacteria, with the exception of C pneumoniae and M pneumoniae, are easily diagnosed by culture-based methods. Indirect serologic methods are the best methods for diagnosing the latter two pathogens.

Although Legionella spp., Mycobacteria spp., and Nocardia spp. can cause pulmonary disease in normal hosts, they may be a more frequent cause of pulmonary disease in immunocompromised hosts. Legionella spp. can be diagnosed by direct examination of pulmonary secretions or alveolar tissue, with a fluorescent antibody technique. Alternatively, acute infection with the most frequently encountered Legionella spp., L pneumophila, can be diagnosed by screening for antigen in the urine. Legionella antigenuria can persist for months after acute infection, a factor that may limit the usefulness of this direct test for diagnosing subsequent L pneumophila infections. Microorganisms that stain poorly by the Gram stain method and that appear to branch or are beaded in appearance should be suspect for Nocardia spp. These organisms frequently stain acid-fast by a modified acid-fast staining method. This method uses less intense decolorizing agents than those used for conventional acid-fast staining of mycobacteria. Nocardia spp. grow more slowly than other bacteria, but can be recovered on standard bacteriologic media. Nocardia spp. also grow well on media used for isolating fungi and mycobacteria and on media used to isolate Legionella spp. (buffered charcoal yeast extract). A recently described opportunist gram-positive bacillus, Rhodococcus equi, also may cause pulmonary infection in immunocompromised patients and, like Nocardia spp., may branch and stain acid-fast by a modified acid-fast staining method.

The dimorphic fungal pathogens (yeast and hyphal forms) H capsulatum, Blastomyces dermatitidis, Coccidioides immitis and Paracoccidioides brasiliensis and the monomorphic fungus (yeast form only) C neoformans may cause pulmonary or disseminated disease in both normal and immunocompromised hosts. The dimorphic fungal pathogen Sporothrix schenckii rarely produces respiratory disease and more frequently presents as cutaneous disease in normal hosts. Monomorphic fungi (hyphal form only) such as Aspergillus spp. generally cause respiratory or disseminated infection in immunocompromised hosts. Pneumocystis carinii has recently been reclassified as a fungus. It is an opportunistic fungal pathogen that causes disease only in immunocompromised hosts, especially patients coinfected with human immunodeficiency virus (HIV). All fungal pathogens, with the exception of P carinii, can be cultured from pulmomary secretions. Diagnosis of P carinii requires direct examination of pulmonary secretions or tissue. Some fungal infections can also be diagnosed by indirect serologic methods; additionally, H capsulatum antigen and C neoformans antigen can be detected in urine and in the serum in disseminated disease.

In certain areas of the world where helminths (Ascaris lumbricoides, Strongyloides stercoralis, Paragonimus westermani, and Echinococcus granulosa) are endemic, pulmonary infection with these parasites may occur. Disseminated infection with S stercoralis, including respiratory infection, may occur in immunocompromised hosts. Toxoplasma gondii may cause pulmonary disease in immunocompromised hosts. Direct examination of wet preps of pulmonary secretions may be useful to identify helminths; direct examination of tissue, culture, and indirect serologic methods are useful for diagnosing T gondii.

Feces

Enteric infections caused by Salmonella spp., Shigella spp., Yersinia enterocolitica, pathogenic E coli, and Campylobacter spp. are increasing in frequency in the United States. These infections are diagnosed by culture of feces, although direct detection of antigens by enzyme-linked immunoassays are sometimes useful for Salmonella spp. and E coli O157:H7. At present, Whipple's disease caused by the bacterium Tropheryma whippelii can be definitively diagnosed by evaluating small-bowel tissue for the presence of nucleic acid that is unique to this organism. Traditionally, periodic acid Schiff staining has been used to demonstrate the organism in tissue (see subsequent discussion).

Occasionally, in severely immunocompromised patients, acid-fast staining and culture may be useful for diagnosing enteric infection caused by Mycobacterium tuberculosis or M avium-intracellulare. However, cultures for mycobacteria should be performed only on stools with positive acid-fast stains. If mycobacteria are recovered from feces, disseminated disease is frequently present.

Enteric parasitic infections are diagnosed by direct examination of fresh or preserved stools; however, direct immunoassays may be useful for some parasitic organisms (Giardia lamblia and Cryptosporidium parvum). Extraintestinal parasitic infections may require other methods, including culture for diagnosis. Not shown in Table 6-2 are culture methods for Trypanosoma spp., Leishmania spp., T gondii, and Entamoeba histolytica. These specialized tests are available only at a few reference laboratories in North America. Culture methods for Acanthamoeba spp. and Naegleria spp. are included in Table 6-1. These methods are easy to perform and especially useful for diagnosing these parasitic infections in patients with corneal infections, which can occur with contact lens use.

Considerable attention has focused recently on newly discovered parasites that are opportunists in immunocompromised patients. These include species of four genera of coccidia (Isospora, Sarcocystis, Cryptosporidium, and Cyclospora) and species of five genera of microsporidia (Enterocytozoon, Septata, Nosema, Encephalitozoon, and Pleistophora). Infections caused by the coccidia or microsporidia have been reported in immunosuppressed patients, notably those patients coinfected with human immunodeficiency virus. Intestinal disease has been demonstrated to occur with all coccidia genera, Enterocytozoon spp., and Septata spp. Extraintestinal disease has been reported with Sarcocystis spp., Cryptosporidium spp., Septata spp., Encephalitozoon spp., Nosema spp., and Pleistophora spp. As previously mentioned, T gondii and the helminth S stercoralis can cause severe disseminated disease in immunocompromised hosts.

Intravascular Catheters

Patients who have indwelling central intravascular catheters for prolonged periods are susceptible to infection. If other sources for infection are ruled out, then infection related to the intravascular catheter must be considered. Diagnosing intravascular-catheter-associated infection can be challenging for the clinician, considering that a definitive diagnosis cannot be achieved unless the catheter is removed and a culture of the tip yields potentially pathogenic bacterium in sufficient quantity (ie, >15 colony forming units of bacteria). If there is evidence for a catheter tunnel infection (subcutaneous infection around the catheter), swabs of the affected area or pus if present should be stained and cultured for bacteria, mycobacteria, and fungi. Occasionally, the fungus Malassezia furfur can infect intravascular catheters and the blood in patients receiving intralipid infusions. Special culture techniques are required to isolate this organism (see Table 6-2).

Surveillance Cultures

In certain instances, surveillance cultures for bacteria and fungi may be useful. Patients who are immunocompromised, receiving broad-spectrum antimicrobial agents, or both may be surveyed for drug resistant bacteria or fungi. As part of infection control programs, institutionalized patients may be surveyed for VRE or methicillin-resistant S aureus carriage. In both of these examples, cultures of the upper airway, feces, or both may be useful to screen for carriage of these potential pathogens.

SEROLOGIC TEST METHODS

Indirect serologic methods may be useful for diagnosing infections caused by certain bacteria, fungi, and parasites and in some cases may be the only means by which a diagnosis is achieved. To diagnose infections by Rickettsia spp., for which alternative diagnostic test methods are limited (attempts at culturing these organisms should be avoided owing to their high infectivity), these tests may be the only means by which a diagnosis is established. Table 6-3 shows the serologic test methods currently available at most reference laboratories. Of note, these methods detect immunoglobulin G (IgG) antibody, IgM antibody, or both to specific bacteria pathogens and for the most part require that the infection has existed in a patient for a finite period so that detectable levels of antibody exist. For IgG analyses, a fourfold increase between baseline and convalescent antibody titers may be required to confirm infection. Because the demonstration of a fourfold rise in antibodies may require > 4 weeks, the diagnostic utility of IgG analyses may be limited, especially in the acute disease phase. However, in some situations, baseline IgG antibody levels may exceed a critical threshold, which is considered diagnostic for infection. Except for the last example, indirect serological methods cannot be considered as rapid diagnostic tests and therefore may be of limited utility. These tests may also be of limited value in patients who lack a humoral response, especially bone marrow transplant recipients.

MOLECULAR TEST METHODS

Molecular test methods, including nucleic acid-probing and -sequencing techniques, allow for the detection of pathogens directly from human specimens. Molecular test methods and nucleic acid amplification techniques, which are frequently available at most reference laboratories are shown in Table 6-5. These methods are potentially useful for fastidious or slowly growing organisms like Legionella spp., Bartonella spp., Mycobacterium spp., Borrelia burgdorferi, and dimorphic fungi. The bacterial agent of Whipple's disease, T whippelii, has never been recovered on culture and presently can be diagnosed only by nucleic acid testing methods. Quantities of parasites in blood or tissue might be sufficiently low (eg, T gondii or Babesia microtii) that direct examination does not provide a diagnosis. In these cases, molecular diagnostic tests may also be useful. It must be emphasized that some studies, particularly those that have evaluated molecular identification methods for group A streptococci and mycobacteria, have demonstrated that these methods, including those that use nucleic acid amplification techniques, are less sensitive than culture. Therefore, if the results for molecular test methods such as these are negative, other diagnostic test methods including culture techniques should be considered.

The National Committee for Clinical Laboratory Standards (NCCLS) provides published guidelines for conventional susceptibility test methods for commonly encountered bacterial organisms that grow aerobically or anaerobically and for yeasts. For bacteria that are not easily cultured (eg, Bartonella spp., Ehrlichia spp., or T whippelii), antimicrobial susceptibility testing is currently not possible. Tentative NCCLS guidelines exist for mycobacteria. No standards exist for parasites. Antimicrobial susceptibility methods include disk diffusion, broth dilution, and agar dilution. For disk diffusion, the inhibition of growth of an organism on solid media is assessed around a paper disk from which an antimicrobial agent diffuses. The greater the zone of inhibition of bacterial growth, the more effective the antimicrobial agent. In the broth dilution procedure, the effect of a known concentration of antimicrobial agent dispersed along with the organism in liquid media is assessed. No growth of the organism (the broth remains clear) indicates that the organism is inhibited at the concentration of antimicrobial agent tested. For agar dilution, a known amount of antimicrobial agent is dispersed in solid medium, and its effect on growth of organisms that are spot inoculated onto the surface of the medium is assessed. No visible growth of the organism means that it is inhibited by the specific concentration of antimicrobial agent that is present in the solid medium.

Interpretation of the results for each of these methods may differ with the antimicrobial agent and organism tested, and guidelines for such are provided by the NCCLS. These interpretations are provided as the following categories: resistant, susceptible, or intermediately susceptible. If an organism is identified as resistant to a particular antimicrobial agent, that agent should not be used in the clinical setting.